Introduction to NGS Ana ConesaHead of Genomics of Gene Expression LabCentro de Investigaciones Prínicpe Felipe firstname.lastname@example.org http://bioinfo.cipf.es/aconesa
Next Generation SequencingNGS has brought high speed not only to genome sequencing and personal medicine, but has also change the way we do genome research: Got a question on genome organization: SEQUENCE IT!!!!
NGS technologies Cost-effective Fast Ultra throughput Cloning-free Short reads
File formatsfastq: sequence data and qualities SAM/BAM: mapping data and qualities
Some Figures How much does it “cost” (computationally) to sequence a human transcriptome? One human transcriptome: 100 Million reads1 Solexa run ==8 lanes ==25 M reads/lane==2 x 4 G fastq/lane (PE) 32 G disk spaceMapping @ processor 12 cores, 48 GB RAM , 4TB disk 24 hoursSAM (Ascii) / BAM (Binary) output 36 G / 9 G
Applications of RNAseqQualitative: Quantitative: * Alternative splicing * Differential expression * Antisense expression * Dynamic range of gene expression * Extragenic expression …. * Alternative 5’ and 3’ usage * Detection of fusion transcripts …. edgeR Tophat/Cufflinks DESeq Scripture baySeq Alexa NOISeq
Advantages of RNAseq? RNAseq microarrays* Non targeted transcript detection * Restricted to probes on array* No need of reference genome * Needs genome knowledge* Strand specificity * Normally, not strand specific* Find novels splicing sites * Exon arrays difficult to use* Larger dynamic range * Smaller dynamic range* Detects expression and SNVs * Does not provide sequence info* Detects rare transcripts * Rare transcripts difficult…. …. and…. are there any disadvantages?????